Human blastoids model blastocyst development and implantation

One week after fertilization, human embryos implant into the uterus. This event requires the embryo to form a blastocyst consisting of a sphere encircling a cavity lodging the embryo proper. Stem cells can form a blastocyst model that we called a blastoid1. Here we show that naive human pluripotent stem cells cultured in PXGL medium2 and triply inhibited for the Hippo, TGF-β and ERK pathways efficiently (with more than 70% efficiency) form blastoids generating blastocyst-stage analogues of the three founding lineages (more than 97% trophectoderm, epiblast and primitive endoderm) according to the sequence and timing of blastocyst development. Blastoids spontaneously form the first axis, and we observe that the epiblast induces the local maturation of the polar trophectoderm, thereby endowing blastoids with the capacity to directionally attach to hormonally stimulated endometrial cells, as during implantation. Thus, we propose that such a human blastoid is a faithful, scalable and ethical model for investigating human implantation and development3,4.

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Software and code
Policy information about availability of computer code Data collection The phase contrast images of cultured cells or cell aggregates were acquired using Thermo Fisher scientific EVOS cell imaging system and inverted wide field microscope Axio VertA1.The fluorescent images and time-lapse images were acquired using Olympus IX83 microscope with Yokogawa W1 spinning disk (Software: CellSense 2.3 ; camera: Hamamatsu Orca Flash 4.0) or Nikon Eclipse Ti E inverted microscope, equipped with a Yokogawa W1 spinning disc (Software: Visiview 4.5.0.7 ; camera:Andor Ixon Ultra 888 EMCCD). Realtime PCR results were collected using CFX384 system (bio-rad). Single cell transcriptome libraries and bulk transcriptome libraries were sequenced using Illumina Novaseq 6000
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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy Raw data for single cell and bulk RNA sequencing data of blastoids were deposited at the GEO repository under the accession number GSE177689. Published human embryo data are available in the GSE109555, E-MTAB-3929.

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Life sciences study design
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Sample size
No statistical methods were used to predetermine sample size. For each experiment, the sample size is determined based on an interval of confidence equal or above 95% as described in the methods and in the text, and on the sampling error, which is estimated based on the standard deviations during pilot studies.
Data exclusions In single cell sequencing data, based on initial evaluation of per-cell quality control metrics and outlier identification using the median absolute deviation algorithm, cells with <= 2000 detected genes or >= 12.5% mitochondrial gene percentage were filtered out.

Replication
All attempts at replication were successful over 3 independent experiments.
Randomization Samples were randomly allocated to groups prior to treatments which prevented any bias in the interpretation of data Blinding The investigators were blinded by preparing in advance the different cocktails and allocating numbers to the different treatments used to stimulate the stem cells.

Eukaryotic cell lines Policy information about cell lines
Cell line source(s) embryonic stem cell lines: Shef6, HNES1 and iPSC lines: cR-nCRM2 and niPSC16.2.b were provided by the laboratory of Austin Smith H9 and H9-GFP reset to naive state were provided by the Laboratory of Yasuhiro Takashima hTSC line bTS5 was provided by the laboratory of Takahiro Arima Endometrial organoids were provided by the laboratory of Hossein Baharvand Authentication H9 (primed and naive PSCs) and TSC-bTS5 were included in single cell sequencing analysis to authenticate their identity

March 2021
Mycoplasma contamination Cells were routinely tested for mycoplasma contamination. No contamination was detected.
Commonly misidentified lines (See ICLAC register) no misidentified lines were used in the study

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Population characteristics
Not applicable because no human subjects were involved in this research. We used donated embryos surplus to IVF treatment and endometrial organoids from a biobank.

Recruitment
Informed consent was obtained from all couples that donated spare embryos following IVF treatment and from people that previously donated endometrial samples that constituted the endometrial biobank used in this study.

Ethics oversight
The use of human embryos donated to research as surplus of IVF treatment was allowed by the French embryo research oversight committee: Agence de la Biomédecine, under approval number RE13-010 and RE18-010. All human preimplantation embryos used in this study were obtained from and cultured at the Assisted Reproductive Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
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Methodology
Sample preparation Cells were collected by dissociated blastoids with sequential treatment of 300units/ml collagenase IV for 30minutes and 0.5% Trypsin (10x) for 20minutes. Cells were stained with Trop2 and PDGFRa antibodies followed by the secondary antibodies Instrument FACS Aria III (BD) Software DiVa version on the F02 is 9.0.1.

Cell population abundance
Abundance of the cell populations of interest was determined by the appropriate negative control and the purity of sorted population was assessed by the post sort analysis.
Gating strategy FSC-A/SSC-A and SSC-H/SSC-W gates were applied to remove debris, and non-single cell aggregates respectively. Dead cells were excluded by using DAPI signal. A example of FACS gating strategy is available at the Supplementary Figure 2A.
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